Fanconi anemia (FA) is an autosomal recessive disorder characterized by pancytopenia, a variety of congenital anomalies, spontaneous chromosome instability, and increased predisposition to malignancy. The molecular basis for the syndrome is unknown, although there is some evidence for defective DNA repair in FA cells. We have recently demonstrated that FA homozygous and heterozygous skin fibroblasts and peripheral blood lymphocytes have increased sensitivity, compared with normal cells, to the clastogenic effect of a nontoxic concentration of the difunctional alkylating agent diepoxybutane (DEB). Variability in both the clinical and cellular manifestations of the disorder is well recognized and strongly suggests an underlying genetic heterogeneity. The objective of this project is to characterize this heterogeneity. Two approaches will be used: (1) We will perform complementation analysis in euploid somatic cell hybrids between fibroblasts from different FA patients. Hybrid cells will be identified cytogenetically in mass culture, using sex chromosome difference in the case of opposite-sex fusions and fluorescent chromosomal heteromorphisms in the case of like-sex fusions. Chromosome breakage will be examined in FA hybrid cells, after exposure of the cells to DEB. A decrease in the extraordinary sensitivity to the clastogenic effect of DEB will be taken as evidence for genetic complementation. (2) We will characterize the clinical and cellular heterogeneity in FA and determine whether these are correlated with genetic differences as demonstrated by the complementation studies. Cellular heterogeneity will be studied by analysis of the level of response of peripheral blood lymphocytes and skin fibroblasts to the clastogenic effect of DEB.